Method to Suppress the Systemic Toxicity of Chemotherapeutic Drugs

ABSTRACT

This invention relates to the combination of manuka honey with other chemotherapeutic agents to treat cancer and reduce the toxicity of the treatment. The invention relates also to an intravenous pharmaceutical composition comprising a diluent; at least 50% w/v of manuka honey diluted by the diluent; and at least one chemotherapeutic agent selected from the group consisting of paclitaxel 5 mg/kg of a patient to be treated; paclitaxel 8 mg/kg of a patient to be treated; oxaliplatin 6 mg/kg of a patient to be treated; and any pharmaceutically acceptable salts thereof. The invention relates further to a method of treating skin cancer, colon cancer, colorectal cancer and/or breast cancer in a patient comprising administering the intravenous pharmaceutical composition to the patient.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. application Ser. No.14/042,818, filed on Oct. 1, 2013, which claims priority from U.S.Provisional Application No. 61/757,096, filed on Jan. 26, 2013, and thecontents of both applications are incorporated herein by reference intheir entirety.

FIELD OF THE INVENTION

This invention relates to a method to suppress the toxicity associatedwith chemotherapeutic drugs for patients subjected to such treatment.The invention also relates to methods to administer the chemotherapeuticdrug in combination with manuka honey in prescribed combination. Thecombination treatment of manuka honey plus chemotherapeutic drugmaintains effective anti-cancer therapy while simultaneously decreasingnonspecific drug toxicity. The invention also relates to a method totreat cancer comprising administering manuka honey.

BACKGROUND OF THE INVENTION

It is becoming a conventional therapy to treat solid tumors withchemotherapeutic drugs. Most of such drugs are associated withtoxicities that affect the well being of patients subjected to suchtherapy. For example, despite the fact that paclitaxel, also known asTaxol, is one of the most potent and effective chemotherapeutic agentsin the treatment of solid tumors, including stage 4 disease, a majordrawback of the drug is its toxic effects on the bone marrow. In somecases it may also cause an anaphylactic shock, which may be fatal inspite of prophylactic hydrocortisone, and benadryl IV. Common sideeffects of paclitaxel include nausea and vomiting, loss of appetite,change in taste, thinned or brittle hair, joint pain in arms or legs,changes in color of nails, tingling in the hands or toes. More seriousside effects include unusual bruising or bleeding, pain, redness andswelling at the injection site, changes in normal bowel habit, fever,chills cough, sore throat, difficulty swallowing, dizziness, shortnessof breath, severe exhaustion, skin rash, facial flushing, femaleinfertility by ovarian damage, and chest pain. Clinical toxicity ofpaclitaxel is associated with the solvent Cremophor EL in which it isdissolved for delivery.

Attempts at reducing toxicity of Taxanes, the class of medicines thatincludes compounds such as paclitaxel and docetaxel, have so far failed.Abraxane is an example; Abraxane (nab-paclitaxel) is paclitaxel bound toalbumen nanoparticles. Abraxis BioScience developed Abraxane to reducetoxicity of paclitaxel by replacing the toxic solvent delivery methodwith Albumen. But clinical trials failed to show any advantage. Thetoxicity of paclitaxel is such that patients taking the drug are betteroff sleeping overnight in hospital. Mortality is high from bone marrowdepression with low WBC with resulting septicemia and irreversibleshock.

Honey has been used for more than 2000 years as traditional medicine indifferent cultures, particularly for its wound healing properties. Theantimicrobial properties of honey have been well described in theliterature. Intrinsic properties of honey like high osmolarity andacidity, as well as the presence of flavonoids and phenolic acids areresponsible for its antibacterial and antioxidant activities (Weston etal., Food Chem 64: 295-301, 1999). In addition to its antimicrobial,antioxidant and tissue-protective activities, recent reports havehighlighted multiple roles for honey in enhancing immune responses,including the induction of inflammatory cytokine production bymacrophages (Tonks et al. Cytokine 21: 242-247, 2003), stimulation ofneutrophil migration (Fukuda et al. Evid Based Complement Alternat Med.2009) and enhanced antibody production (Al-Waili, J Med Food 7: 100-107,2004). Whether the multitude of honey activities is mediated by the sameor different active fractions remains to be fully elucidated.

Manuka honey, obtained from nectar collected by honey bees (ApisMellifera) from the New Zealand manuka tree (Leptospermum scoparium), isa complex mixture of carbohydrates, fatty acids, proteins, vitamins andminerals containing various kinds of phytochemicals with high phenolicand flavonoid content (Yao et al. Food Chemistry 81: 159-168, 2003).While manuka honey shares constituents, e.g. glucose-oxidases, withother honeys it also contains other phytochemical factors thatpotentiate its antibacterial activity like methylglyoxal (Mavric et al.,Mol Nutr Food Res 52: 483-489, 2008). This gave rise to a classificationsystem adopted for active manuka honey, known as unique manuka factor(UMF), an indication of its antibacterial activity (Allen et al. J PharmPharmacol 43: 817-822, 1991).

Previous studies addressing the mechanisms of the anti-bacterialactivity of manuka honey identified a number of potential activeconstituents, including several phenolic compounds that act asscavengers of superoxide anion radicals (Inoue et al., J Sci Food Agri85: 872-878, 2005, Jenkins et al, Jenkins et al. Int J Antimicrob Agents37: 373-376, 2011, Kawakman et al, PLoS One 6: e17709, 2011). There isevidence that the antibacterial activity of manuka honey is independentof its role in inducing inflammatory cytokines during innate immuneresponses.

A 5.8 kD, heat-sensitive, protease-resistant, component, that was devoidof any antibacterial activity was identified to be responsible for theinduction of cytokine production via interaction with TLR4 onmacrophages (Tonks et al. J Leukoc Biol 82: 1147-1155. 2007). Thesestudies suggest the presence of unique, yet-to-be-characterized,constituents with desired activities in manuka honey. However, aninvestigation of the anti-proliferative properties of manuka honey hasnot been undertaken.

Perhaps, one of the oldest known uses for honey is in wound healing.There is extensive scientific and clinical evidence to support theutilization of honey for wounds, skin reactions and damage to epithelialbarriers following radiotherapy and chemotherapy (Bandy et al, J ClinNurs 17: 2604-2623 2008). In patients with chronic wounds or burns,honey has been shown to stimulate angiogenesis and epithelialization,promoting more efficient healing (Molan Am J Clin Dermatol 2: 13-192001, Wijesinghe N Z Med J 122: 47-60, 2009). More recently, severalreports demonstrated that honey, being rich in polyphenols andflavonoids, has anti-proliferative effects against cancer cells(Jaganathan et al. J Biomed Biotechnol 2009: 830616, 2009, Ghashm et al.BMC Complement Altern Med 10: 49, 2010, Swellam et al. Int J Urol 10:213-219, 2003). However, the mechanisms for the anti-cancer effect arestill to be fully elucidated. In an early study, honey was shown toexhibit modest anti-tumor, but good anti-metastatic, activities againsta number of tumor cell lines (Gribel & Pashinskii Vopr Onkol 36:704-709,1990). Another study extended this observation and showed that dietaryintake of caffeic acid esters, a major constituent of Propolis honeybeehives, inhibited the incidence and multiplicity of invasive andnon-invasive carcinogen-induced colon adenocarcinomas (Rao et al CancerRes 53:4182-4188, 1993). More recently, diluted unfractionated honey wasshown to inhibit the proliferation of bladder cancer cell lines invitro. Moreover, intralesional injection of honey was found to inhibittumor growth in a bladder cancer implantation mouse model, but theeffect of the treatment on animal survival was not reported (Swellam etal. Int J Urol 10: 213-219, 2003).

The effect of manuka honey on the growth of cancer cells, using both invitro as well as in vivo approaches, was investigated. The findingsprovide mechanistic evidence for the induction of apoptosis in cancercells by manuka treatment and further highlight a novel role forsystemically-administered manuka honey as both an anti-cancer agent andan adjuvant in combination with standard chemotherapeutic agents.Moreover, the data provides direct evidence that manuka honeyadministration reverses the systemic toxicity associated with the use ofa chemotherapeutic agent, such as paclitaxel and/or Oxaliplatin.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating cancer in a patientcomprising administering to the patient a composition comprising manukahoney.

The invention further relates to methods of treating cancer comprisingadministering to a patient a chemotherapeutic agent and a compositioncomprising manuka honey.

Another aspect of the invention relates to methods to maintainanti-oxidant levels in a patient during treatment with achemotherapeutic agent comprising administering a composition comprisingmanuka honey to the patient.

Other aspects of the invention also relate to methods to reduce thetoxicity of a chemotherapeutic agent in a patient comprisingadministering a composition comprising manuka honey to the patient.

A further aspect relates to a method of reducing the dose of achemotherapeutic agent administered to a patient during cancertreatment, comprising administering the chemotherapeutic agent withmanuka honey.

Preferably the further chemotherapeutic agent is a taxene orOxaliplatin. Oxaliplatin is a synthetic platinum-based chemotherapeuticdrug which is currently licensed for use in advanced colorectal as wellas colon cancers. Based on some studies, it has also shown activityagainst metastatic breast cancer.

In a particular embodiment, the chemotherapeutic agent is paclitaxel(taxol), or its pharmaceutically acceptable salt.

Preferably the manuka honey composition is in the form of a solutioncomprising from about 10% w/v to about 60% w/v manuka honey. Preferablythe composition comprises between 20-50% w/v manuka honey. Thecompositions can comprise 20%, 25%, 30%, 35%, 40%, 45%, or 50% w/vmanuka honey.

Suitable forms for the manuka honey include pharmaceutical forms forparenteral administration, in particular forms for administering themanuka honey intravenously.

The manuka honey composition can be administered to the patientsequentially or simultaneously with the chemotherapeutic agent. Whenadministered simultaneously the chemotherapeutic agent and the manukahoney can be in the same or separate compositions.

The manuka honey composition and the chemotherapeutic agent arepreferably administered intravenously.

In some embodiments the manuka honey and the chemotherapeutic agent canbe administered sequentially. The manuka honey and the chemotherapeuticagent can be administered at least 12 hours apart, at least 1 day apart,at least 2 days apart or at least 3 days apart. In other furtherembodiments the manuka honey and the chemotherapeutic agent areadministered together.

The methods of the present application are useful for the treatment ofvarious diseases, including, for example, breast cancer, lung cancer(such as small cell lung cancer and non-small cell lung cancer), renalcancer, bladder cancer, pancreatic cancer, ovarian cancer, prostatecancer, brain cancer, colorectal cancer, leukemia, lymphoma, multiplemyeloma and solid tumors. The methods are particularly suitable fortreating skin, colon, ovarian, colorectal, lung and/or breast cancers.In one embodiment the methods are suitable for treating small cell lungcancer. In further embodiments the methods are suitable for treatingcolon or colorectal cancer. In a further embodiment the methods aresuitable for treating breast cancer. In a further embodiment the methodsare particularly suitable for treating lung cancer. In a furtherembodiment the methods are particularly suitable for skin cancer. In afurther embodiment the methods are suitable for treating ovarian cancer.

The invention further relates to manuka honey for use in treatingcancer. In particular, the manuka honey is useful in treating, breastcancer, lung cancer (such as small cell lung cancer and non-small celllung cancer), renal cancer, bladder cancer, pancreatic cancer, ovariancancer, prostate cancer, brain cancer, colorectal cancer, leukemia,lymphoma, multiple myeloma and solid tumors. The manuka honey isparticularly useful for treating skin cancer, colon or colorectalcancer, ovarian cancer, colon cancer, lung cancer and/or breast cancer.

The invention further relates to manuka honey for use in combinationwith a chemotherapeutic agent for treating cancer.

A further aspect of the invention relates to manuka honey for use incombination with a chemotherapeutic agent to maintain anti-oxidantlevels in a patient treated with the chemotherapeutic agent.

Another aspect of the invention relates to manuka honey for use incombination with a chemotherapeutic agent to reduce the toxicity of thechemotherapeutic agent in a patient.

A further aspect of the invention relates to manuka honey for use inreducing the dosage of a chemotherapeutic agent administered to apatient. The use comprises administering the manuka honey in combinationwith the chemotherapeutic agent.

In one embodiment the manuka honey is used as an adjunct therapy tochemotherapy. Preferably the manuka honey is for use as an adjuncttherapy to at least one of paclitaxel therapy and Oxaliplatin therapy.

The invention further relates to a pharmaceutical composition comprisinga chemotherapeutic agent and manuka honey. The composition can be acombined preparation for simultaneous or separate use of the manukahoney and the chemotherapeutic agent.

Preferably the pharmaceutical composition is in the form of a solution.Preferably the chemotherapeutic agent and manuka honey are in a formsuitable for intravenous administration.

Preferably the pharmaceutical composition comprises 10% to 60% w/vmanuka honey. In one embodiment the composition comprises from 20 to 50%w/v manuka honey.

The chemotherapeutic agent is preferably selected from the groupconsisting of paclitaxel, Oxaliplatin and pharmaceutically acceptablesalts thereof.

The invention also relates to the use of the manuka honey to treatcancer. One embodiment comprises the use of manuka honey whenadministered with a chemotherapeutic agent to treat cancer.

The invention also relates to the use of manuka honey and achemotherapeutic agent in the treatment of cancer.

The invention further relates to the use of manuka honey to reduce thedose of the chemotherapeutic agent given to a patient.

Preferably the invention relates to the use of manuka honey incombination with one of paclitaxel and oxaliplatin.

According to a particular aspect of the invention there is provided anintravenous pharmaceutical composition comprising:

a diluent;

at least 50% w/v of manuka honey diluted by the diluent; and

at least one chemotherapeutic agent, wherein the chemotherapeutic agentis selected from the group consisting of paclitaxel between about 5mg/kg of a patient to be treated and 10 mg/kg of a patient to betreated; oxaliplatin at least 1 mg/kg of a patient to be treated; andany pharmaceutically acceptable salts thereof.

In a particular embodiment, the chemotherapeutic agent is selected fromthe group consisting of paclitaxel 5 mg/kg of a patient to be treated,paclitaxel 8 mg/kg of a patient to be treated and oxaliplatin 6 mg/kg ofa patient to be treated.

The pharmaceutical composition may be in the form of a solution. Thediluent may be a sterile saline solution.

According to another aspect of the invention there is provided a methodof treating skin cancer, colon cancer, colorectal cancer and/or breastcancer in a patient comprising administering to the patient apharmaceutically acceptable amount of a composition as described anddefined hereinabove.

The manuka honey and the chemotherapeutic agent may be administeredsequentially. The manuka honey and chemotherapeutic agent may beadministered simultaneously. The method may be for enhancing thesurvival of the patient.

The method may further comprise administered a second chemotherapeuticagent. The second chemotherapeutic agent may be selected from the groupconsisting of cisplatin, doxorubicin, trastuzumab, and anypharmaceutically acceptable salts thereof.

The method may be for reducing the toxicity of said at least onechemotherapeutic agent in the patient. The method may be for reducingthe toxicity of the second chemotherapeutic agent.

According to another aspect of the invention there is provided a unitdose or multi-dose container containing the intravenous pharmaceuticalcomposition as described and defined hereinabove.

The term “treatment” is intended to include curing, reversing,alleviating, palliative and prophylactic treatment of the condition.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Combination therapy encompassesco-administration of these therapeutic agents, in a substantiallysimultaneous manner, either in the same composition or in separatecompositions independently administered.

In one embodiment the manuka honey and the chemotherapeutic agent areformulated into the same composition at an appropriate finalconcentration for administration to the patient together in oneinjection.

In some embodiments the administration of the chemotherapeutic agent andthe manuka honey composition is concurrent, i.e. the administrationperiod of the chemotherapeutic agent and the administration period ofthe manuka honey composition overlap. In some embodiments concurrentadministration includes starting the administration of thechemotherapeutic agent and the manuka honey composition at the sametime. In some embodiments concurrent administration involves startingthe administration of a second composition after the administration of afirst composition has started.

In addition combination therapy encompasses co-administration of eachtype of therapeutic agent in a sequential manner. Sequentialadministration of the chemotherapeutic agent and the manuka honeycomposition can involve administration of the manuka honey compositionprior to or after the administration of the chemotherapeutic agent. Insome embodiments the administration of the chemotherapeutic agent andthe manuka honey composition are non-concurrent. For example theadministration of one of the composition is finished before theadministration of the other agent begins.

In some embodiments there may be a therapeutically effective timeinterval between the administration of the chemotherapeutic agent andthe administration of the manuka honey composition. In some embodimentsthe time interval between the start of the administration of one of thecomposition and the start of the administration of the other compositionmay be about 0.5, 1, 2, 3, 4 or more days. In some embodiments theadministration of the first composition is finished beforeadministration of the second composition begins. In certain embodimentsthe administration of the first composition continues when theadministration of the second composition begins.

Patients suffering from cancer are commonly co-administered additionaltherapeutic agents, in particular anti-neoplastic and/or furtheranti-tumor agents. Therefore the invention further relates to acombination therapy of manuka honey and at least one of paclitaxel andOxaliplatin, in combination with one or more further suitable anti-tumoragents for the treatment of cancer. Further suitable anti-tumor agentsinclude for example cisplatin, doxorubicin, trastuzumab. Other suitableagents are also encompassed.

Other therapeutic agents are also commonly administered to patients todeal with the side effects of chemotherapy. Such agents might includeanti-emetics for nausea, or agents to treat anaemia & fatigue. Othersuch medicaments are well known to physicians and others skilled incancer therapy.

The manuka honey may be formulated for parenteral administration byinjection. The compositions may take forms such as suspensions orsolutions. The formulations may be presented in unit-dose or multi-dosecontainers. The compositions may include additional ingredients that areconventional in the art with regard to injectable formulations. Methodsof preparing various pharmaceutical compositions are well known to thoseskilled in the art. Reference is made to ‘Remington's PharmaceuticalSciences’.

In one embodiment the manuka honey can be formulated at the appropriateconcentration in a sterile saline solution.

The actual amount of the compositions to be administered, the rate ofadministration and the time period of administration will depend on anumber of factors. These include, for example, the type of cancer to betreated, the chemotherapeutic agent to be used, and the size, location,progression and/or severity of the cancer to be treated. Appropriatedosage and regimes for chemotherapeutic agents such as paclitaxel, arewell known in the art.

In some embodiments paclitaxel is administered at a dose of about 75 to250 mg/m² (˜2.0 to 6.8 mg/kg) over a period of time. The usual dosage ofpaclitaxel is 135-225 mg/m² (3.6-6.0 mg/kg) given intravenously over 3hours every 2-3 weeks. In a further embodiment paclitaxel isadministered at a reduced dosage.

In other embodiments Oxaliplatin is administered at a dose of about 6mg/kg over a period of time. The usual dosage of Oxaliplatin is 6 mg/kgadministered intraperitoneally (i.p.) once per week for a period ofthree weeks.

In humans with colorectal cancer, oxaliplatin is administeredintravenously in combination with other drugs at a dose of ˜2 mg/kg onceevery 2 weeks. This cycle is repeated for up to 12 times depending onpatient condition and response.

By a reduced dosage of a chemotherapeutic agent it is meant providingthe chemotherapeutic in a dosage less than the usual dosage of thechemotherapeutic agent when administered without manuka honey. Forexample, providing paclitaxel to the patient in a dose smaller than theusual dosage that would otherwise be administered to the patient in theabsence of manuka honey during therapy.

The concentration of the manuka honey is typically in the range from 10%w/v to 60% w/v in an aqueous solution. In further embodiments theconcentration is in the range from about 10% w/v to about 50% w/v, about20% to about 50% w/v, and more preferably from about 30% w/v to about50% w/v. In some embodiments the methods for treating cancer involveinfusing the manuka honey composition once every three days. In someembodiments 75-200 ml of a manuka honey composition is infused twice perweek, once weekly, once biweekly, or once tri-weekly. Preferably 100 mlof the manuka honey composition is infused with each treatment roundprovided to the patient.

In some embodiments the method for treating cancer involvesintravenously administering about 75 to about 250 mg/m² of paclitaxeland administering a composition comprising manuka honey at aconcentration of 10% to 60% w/v.

Disclosed are combination treatments of paclitaxel with manuka honeythat result in a significant improvement in overall animal survival.

The use of manuka honey in combination with paclitaxel has beendemonstrated by various in vitro and in vivo models. Althoughtumor-bearing mice treated with paclitaxel alone exhibited a 60%reduction in tumor growth, their overall survival was only 20%.Tumor-bearing animals treated with manuka honey plus paclitaxelexhibited a similar degree of inhibition in their tumor growth but, insharp contrast, there was a 3-fold enhancement in animal survival.

Animal toxicity studies, utilizing multiple intravenous administrationsof manuka honey, confirmed that systemic administration of manuka honeyis not associated with any systemic toxicity. Analysis of hematological(total WBC, neutrophils, lymphocytes, monocytes, RNC and platelets) aswell as clinical chemistry (ALT, AST, LDH, creatinine, BUN, and glucose)parameters in manuka honey-injected animals showed no alterations fromnormal controls. Administration of paclitaxel to animals resulted in asignificant decrease in the level of antioxidant in various organs.However, simultaneous treatment with paclitaxel plus uniqueconcentrations of manuka honey was found to reverse the taxol-induceddeleterious effects on organ anti-oxidant levels.

The combination of manuka with paclitaxel is effective in reducingoverall toxicity, leading to improved animal survival and effectivecontrol of tumor proliferation. This combination of effects, namelyeffective control of cancer growth and reduced tissue toxicity, maypotentially represent a breakthrough in cancer treatment.

Manuka honey has been recognized for its anti-bacterial andwound-healing activity but its potential antitumor effect is poorlystudied despite the fact that it contains many antioxidant compounds.Disclosed is a combination that demonstrates anti-proliferative activityof manuka honey on three different cancer cell lines in vitro, murinemelanoma (B16.F1) colorectal carcinoma (CT26) and human breast cancer(MCF-7) cells. The invention demonstrates that manuka honey has potentanti-proliferative effect on all three cancer cell lines in a time anddose-dependent manner, being effective at concentrations as low as 0.6%(w/v). Without being bound by any hypothesis, it is believed that thiseffect is mediated via the activation of a caspase 9-dependent apoptoticpathway, leading to the induction of caspase-3, reduced Bcl-2expression, DNA fragmentation and cell death. Combination treatment ofcancer cells with manuka plus paclitaxel in vitro, however, revealed noevidence of a synergistic action on cancer cell proliferation.Furthermore, an in vivo syngeneic mouse melanoma model was utilised toassess the potential effect of intravenously administered manuka honey,alone or in combination with paclitaxel, on the growth of establishedtumors. The combination indicates that systemic administration of manukahoney was not associated with any alterations in haematological orclinical chemistry values in serum of treated mice, demonstrating itssafety profile. Treatment with manuka honey alone resulted in about 33%inhibition of tumor growth, which correlated with histologicallyobservable increase in tumor apoptosis. Although better control of tumorgrowth was observed in animals treated with paclitaxel alone or incombination with manuka honey (61% inhibition), a dramatic improvementin host survival was seen in the co-treatment group. This highlights arole for manuka honey in alleviating chemotherapy-induced toxicity. Insummary, the findings demonstrate that:

(1) Manuka honey is able to inhibit the growth of different cancer celllines (e.g. human breast cancer, murine colon cancer and murinemelanoma) in vitro, even at exceedingly low concentrations (<1%).

(2) Manuka exerts its anti-cancer activity via the activation of caspase9-dependent, intrinsic, apoptosis pathway. This is in sharp contrast topaclitaxel that induces cancer cell death via the activation of acaspase 8-dependent, extrinsic apoptosis pathway.

(3) When used in an experimental animal melanoma model, the combinationof manuka plus paclitaxel resulted in effective inhibition of tumorgrowth and a marked enhancement in animal survival.

(4) When used in combination with paclitaxel, manuka reverses systemic,chemotherapy associated, organ toxicities.

In another embodiment, the method of treating cancer involvesintravenous administration of about 1 mg/kg to about 6 mg/kg, preferablyabout 6 mg/kg of Oxaliplatin and administering a composition comprisingmanuka honey at a concentration of 10% to 60% w/v.

In another embodiment, the method of treating cancer involvesintravenous administration of about 1 mg/kg to a maximumpharmaceutically acceptable amount of Oxaliplatin and administering acomposition comprising manuka honey at a concentration of 10% to 60%w/v.

Disclosed are combination treatments of Oxaliplatin with manuka honeythat result in a significant improvement in overall animal survival.

The use of manuka honey in combination with Oxaliplatin has beendemonstrated by various in vitro and in vivo models. More specifically,combination treatment with Manuka honey and Oxaliplatin has been foundto be effective in inhibiting colon cancer and breast cancer inexperimental mouse models.

Treatment with a combination of manuka honey with Oxaliplatin has beenfound to result in effective inhibition of colon cancer growth.Treatment with a combination of manuka honey with Oxaliplatin hasfurther been found to result in a significant enhancement in hostsurvival.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate embodiments of the presently disclosedinvention and, together with the description, disclose the principles ofthe invention.

FIGS. 1A-1G demonstrates the ability of manuka honey to inhibit cancercells. Skin cancer cells (B16.F1 cell line) (FIGS. 1A-1C), colon cancercells (CT26 cell line) (FIGS. 1D, 1E) and breast cancer cells (MCF-7cell line) (FIGS. 1F, 1G) were plated at 5×103 cells per well andincubated for 24 hr (FIGS. 1A, 1D, 1F), 48 hr (FIG. 1B) or 72 hr (FIGS.1C, 1E, 1G) in the absence or presence of the indicated concentrationsof manuka honey (range 0.3% to 5.0% w/v), or taxol (10 ng/ml or 50 ng/mlfinal concentration). At the end of the incubation period, cellviability was determined using CellTiter-Glo luminescent assay. Resultsare expressed as percentage viability in treated cell cultures comparedto control, untreated, cells and are representative of 3 (for B16.F1cells) or 2 (for CT26 and MCF-7 cells) independent experiments.Asterisks denote statistically significant differences in viability ofexperimental groups compared to control (*, p<0.05; **, p<0.01; ***,p<0.001).

FIGS. 2A-2B shows that Manuka honey induces the death of cancer cells byan apoptotic mechanism. B16.F1 cells were treated for 24 hr (leftcolumn), 48 hr (center column) or 72 hr (right column) with varyingconcentrations of manuka honey (M; range 0.3%-5.0%), taxol (10 ng/ml) ormedium as control. At the end of the incubation period, cells wereharvested and stained with Annexin V and PI, and analyzed byflowcytometry. The percentages of cells in early (Annexin V+, PI−; lowerright quadrant) and late apoptotic-necrotic stages (Annexin V+, PI+;upper right quadrant) are shown. The results are representative of threeindependent experiments.

FIGS. 3A-3C demonstrates that manuka honey induces the death of cancercells by activating the intrinsic pathway of caspase-mediated apoptosis.B16.F1 melanoma cells were treated with manuka honey (5% w/v), taxol (10or 50 ng/ml) or medium as control. After 24 hr of culture, enzymaticactivity of caspase 3/7 (FIG. 3A) and caspase 8 (FIG. 3B) weredetermined using specific kits and following manufacturer'srecommendation. The data is presented as fold increase in caspaseactivity after normalization to the number of viable cells per culture.FIG. 3C. Western blot analysis of caspase-9 activation B16.F1 cellstreated with manuka honey or taxol. Whole cell extracts were preparedafter a 24-hr treatment with manuka honey (5% w/v) or taxol (10 ng/ml).Protein extracts were resolved on 10% SDS-PAGE and immunoblotted withcaspase-9-specific ployclonal antibody capable of detecting both fulllength and cleaved forms of caspase-9. The cell extracts were alsoprobed with an antibody against β-actin as a control for proteinloading.

FIGS. 4A-4C. Manuka honey induces late apoptotic events in cancer cells.FIG. 4A. B16.F1 cells were incubated for 24 hr or 72 hr in the absenceor presence of Manuka honey (M; 5%) or taxol (T; 50 ng/ml). Whole cellextracts (100 μg/lane) were resolved on 10% SDS-PAGE followed by Westernblotting with an antibody specific to Bcl-2. FIG. 4 B. Cells weretreated for 24 hr or 72 hr with the indicated concentrations of manukahoney (0.6%-5.0%) or taxol (50 ng/ml). Whole cell extracts (100 μg/lane)were resolved on 10% SDS-PAGE followed by Western blotting with aPARP-specific antibody. The full-length (116 kD) and cleaved (89 kD)forms of PARP are indicated. The cell extracts were also probed with anantibody against β-actin as a control for loading. FIG. 4C. Followingtreatment for 72 hr, cells were lysed and DNA extracted bycentrifugation and phenol-chloroform extraction. Extracted DNA wasresolved on 1.5% agarose gel and stained with ethidium bromide tovisualize the oligonucleosomal fragments. The results are representativeof two independent experiments.

FIGS. 5A-5F and FIGS. 6A-6F illustrate that systemic administration ofmanuka honey is not associated with any alterations in hematologicalvalues (FIG. 5) or clinical chemistry parameters (FIG. 6). Mice wereinjected with saline or manuka honey (50% w/v) 2 times per week for atotal of 3 weeks, following which blood was collected and analyzed forthe indicated parameters. In each graph, the values for individual micein a group are shown, together with the mean±SEM. The shaded box in eachgraph represents the normal range for that particular parameter. Theresults are representative of three independent experiments.

FIGS. 7A-7B show the results from the systemic administration of manukahoney at different concentrations on tumor growth and animal survival.Manuka honey at either 5%, 10%, or 20% w/v, or saline were administeredto the animals twice per week until the end of the observation period.

FIGS. 8A-8B shows that systemic administration of manuka honey reducestumor growth and enhances animal survival. (FIG. 8A) Animals withestablished tumors were treated i.v. with either manuka honey (50% w/v),taxol (10 mg/Kg), manuka honey+taxol, or saline as control. Alltreatments were given twice per week until the end of observationperiod. Each data point represents the mean±SEM of 19-20 mice per group,pooled from 2 individual experiments. Asterisks denote statisticallysignificant differences between each experimental group and the salinecontrol group; also shown is a comparison between manuka honey alone andmanuka honey+taxol groups (**, p<0.01; *** p<0.001). (FIG. 8B)Co-treatment with taxol and manuka honey leads to a significantenhancement in host survival. Experimental animals were followed forsurvival for up to day 25 post treatment. Each data point represents themean±SEM of 19-20 mice per group, pooled from 2 individual experiments.Asterisks denote statistically significant differences betweenexperimental and saline control groups; also shown is a comparisonbetween taxol alone and manuka honey+taxol groups (**, p<0.01; *,p<0.05).

FIG. 9 shows that systemic administration of manuka honey and taxolreduces tumor growth and enhances animal survival. Animals withestablished tumors were treated i.v. with either manuka honey (50% w/v),taxol (5 mg/Kg), manuka honey+taxol, or saline as control. Alltreatments were given twice per week until the end of observationperiod. Asterisk denotes statistically significant differences betweenthe saline control group and manuka honey +taxol group (*, p<0.05).

FIGS. 10A-10C show that systemic administration of manuka honey andtaxol reduces tumor growth (FIG. 10A and FIG. 10C) and enhances animalsurvival (FIG. 10B). Animals with established tumors were treated i.v.with either manuka honey (50% w/v), taxol (5 mg/Kg), manuka honey+taxol,or saline as control. All treatments were given twice per week until theend of observation period. (FIG. 10A) Kinetics of tumor growth in thedifferent experimental groups. (FIG. 10B) Host survival was followed forup to day 25 post treatment. (FIG. 10C) Comparison of tumor volumes inthe four experimental animals groups at day 17 post treatment. In allpanels, asterisks denote significant differences (*, p<0.05; **,p<0.01).

FIGS. 11A-11E illustrates the increase in the ratio of caspase3-positive (apoptotic) tumor cells by direct immunohistochemicalstaining following treatment with the combination of manuka honey pluspaclitaxel. Tumor tissue sections were prepared after treatment withsaline (FIG. 11A), manuka honey (FIG. 11B), paclitaxel (FIG. 11C) ormanuka honey+paclitaxel (FIG. 11D) and stained using caspase 3-specificantibody. Representative images at high magnification (bar=50 μm) areshown. Arrows indicate representative, brown-staining, apoptotic cells.Necrotic regions are also indicated (n). The results are representativeof two independent experiments. (FIG. 11E) Quantitative estimation ofthe number of caspase-3 positive cells in tumor sections of differenttreatment groups. The data is shown as the mean±SEM of the number ofpositive cells per high power field. Tumors were obtained from 2-3 miceper treatment group and multiple sections were made from each tumortissue. The number of positive cells was determined by counting thenumber of cells in 20 high power fields per section. Asterisks denotestatistically significant differences between each experimental groupand the saline control group; also shown is a comparison between manukahoney alone and manuka honey+taxol groups (*, p<0.05).

FIGS. 12A-12C desmonstrate that manuka honey reverses the systemic,toxic, side-effect of paclitaxel. Animals were treated with saline,paclitaxel, manuka honey or manuka honey plus paclitaxel. At the end ofa 3-week treatment, animals were sacrificed and organs removed fortoxicity determination. The levels of glutathione (GSH) in the liver(FIG. 12A) spleen (FIG. 12B) and kidney (FIG. 12C) were determined. Thedata represent the mean±SEM of a minimum of 3 determinations perexperimental group. For all panels, asterisks denote significantdifferences between the indicated groups (*, p<0.05; **, p<0.01; ***,p<0.001).

FIG. 13 shows a graph showing change in tumor volume over a period of 21days in melanoma-bearing mice treated with paclitaxel (5 mg/kg) alone;saline (control); manuka honey alone (50% w/v) and a combination ofpaclitaxel (5 mg/kg) and manuka honey alone (50% w/v), asterisks denotestatistically significant differences between the indicated experimentalgroups (**, p<0.01; *, p<0.05);

FIG. 14 shows a table showing change in tumor volume at day 21 inmelanoma-bearing mice treated with paclitaxel (5 mg/kg) alone; saline(control); manuka honey alone (50% w/v) and a combination of paclitaxel(5 mg/kg) and manuka honey (50% w/v);

FIG. 15 shows a graph showing percentage survival over a period of 25days in melanoma-bearing mice treated with paclitaxel (5 mg/kg) alone;saline (control); manuka honey alone (50% w/v) and a combination ofpaclitaxel (5 mg/kg) and manuka honey (50% w/v), asterisks denotestatistically significant differences between the indicated experimentalgroup in comparison to the control group (*, p<0.05);

FIG. 16 shows a table showing percentage survival at day 25 inmelanoma-bearing mice treated with paclitaxel (5 mg/kg) alone; saline(control); manuka honey alone (50% w/v) and a combination of paclitaxel(5 mg/kg) and manuka honey (50% w/v);

FIG. 17 shows a graph showing change in tumor volume over a period of 17days in melanoma-bearing mice treated with paclitaxel (8 mg/kg) alone;saline (control); manuka honey alone (50% w/v) and a combination ofpaclitaxel (8 mg/kg) and manuka honey (50% w/v), asterisks denotestatistically significant differences between the indicated experimentalgroups (**, p<0.01; *, p<0.05);

FIG. 18 shows a table showing change in tumor volume at day 17 inmelanoma-bearing mice treated with paclitaxel (8 mg/kg) alone; saline(control); manuka honey alone (50% w/v) and a combination of paclitaxel(8 mg/kg) and manuka honey (50% w/v);

FIG. 19 shows a graph showing percentage survival over a period of 25days in melanoma-bearing mice treated with paclitaxel (8 mg/kg) alone;saline (control); manuka honey alone (50% w/v) and a combination ofpaclitaxel (8 mg/kg) and manuka honey (50% w/v), asterisks denotestatistically significant differences between the indicated experimentalgroups (*, p<0.05);

FIG. 20 shows a table showing percentage survival at day 25 inmelanoma-bearing mice treated with paclitaxel (8 mg/kg) alone; saline(control); manuka honey alone (50% w/v) and a combination of paclitaxel(8 mg/kg) and manuka honey (50% w/v);

FIG. 21 shows a graph showing percentage survival over a period of 49days in colon cancer-bearing mice treated with oxaliplatin (6 mg/kg)alone; saline (control); manuka honey alone (50% w/v) and a combinationof oxaliplatin (6 mg/kg) and manuka honey (50% w/v), Asterisks denotestatistically significant differences between the indicated experimentalgroups (*, p<0.05);

FIG. 22 shows a table showing percentage survival at day 49 in coloncancer-bearing mice treated with oxaliplatin (6 mg/kg) alone; saline(control); manuka honey alone (50% w/v) and a combination of oxaliplatin(6 mg/kg) and manuka honey (50% w/v);

FIG. 23 shows a graph of tumor volume over a period of 50 days inmetastatic breast cancer-bearing mice treated with oxaliplatin (6 mg/kg)alone; saline (control); manuka honey alone (50% w/v) and a combinationof oxaliplatin (6 mg/kg) and manuka honey (50% w/v), asterisks denotestatistically significant differences between the indicated experimentalgroups (*, p<0.05);

FIG. 24 shows a table showing tumor volume at day 50 in metastaticbreast cancer-bearing mice treated with oxaliplatin (6 mg/kg) alone;saline (control); manuka honey alone (50% w/v) and a combination ofoxaliplatin (6 mg/kg) and manuka honey (50% w/v); and

FIG. 25 shows a graph illustrating the differential gene expression ofpaclitaxel/dextrose vs. paclitaxel/manuka honey.

DESCRIPTION OF THE INVENTION

Despite the remarkable advances made over the past 50 years inunderstanding the basis of cancer development, and the increasedavailability of treatment modalities, cancer-related death toll remainsone of the highest among chronic human diseases. A major concern foranti-cancer drugs is their potential toxicity. Considerable effortscontinue to be exerted to identify naturally occurring compounds, ortheir principle active components, with potential to complement existingcancer therapeutic modalities. The current invention highlights severalfindings regarding the utility of manuka honey as a potentialanti-cancer agent. First, multiple intravenous injections of manukahoney, administered over a period of 2-3 weeks, caused no apparentsystemic side effects, as judged by the results of the hematological andclinical chemistry analyses which showed no alterations in the cellularconstituents of blood or chemical markers of organ dysfunction in theserum of treated animals. Second, manuka honey treatment resulted in asignificant growth inhibition (˜33%) in a melanoma tumor model known forits aggressiveness and low immunogenicity. Third, several lines of invitro evidence demonstrate that manuka honey induces death of cancercells via the activation of caspase-9-dependent intrinsic apoptosispathway. Finally, intravenous co-administration of paclitaxel and manukahoney resulted in a highly significant inhibition of tumor growth andimproved overall animal survival.

In order to demonstrate the invention, experiments were done to studythe physiochemical characteristics of the manuka honey. Differentdilutions of manuka honey were prepared directly in tissue culturemedium in which the B16.F1 melanoma cells are routinely cultured andtested for their pH and osmolarity. The studies revealed that manukahoney solutions of concentrations up to 5% (w/v) were physiological(data not shown). Thus, all subsequent in vitro studies were carried outusing manuka honey concentrations in the range of 0.3% to 5%.

For all experiments paclitaxel (Sigma, St Louis, Mo., USA) was initiallydiluted in sterile saline solution. The drug was further diluted to thedesired final concentration in either further sterile saline for i.v.studies or freshly diluted in culture medium for in vitro studies.Manuka honey (UMF 10+, Honeyland NZ Ltd, New Zealand) was diluted insterile saline or culture medium for in vivo or in vitro studiesrespectively.

Manuka Honey Inhibits Growth of Cancer Cells

The potential effect of manuka honey on cancer cell proliferation wasinvestigated using three tumor cell lines differing in type and origin,the murine melanoma (B16.F1) and colon carcinoma (CT26) cells and thehuman breast cancer (MCF-7) cell line. Cells were incubated withdifferent concentrations of manuka honey (range 0.3 to 2.5% w/v) for24-72 hrs. As a positive control, cells were cultured with taxol at afinal concentration of 10 or 50 ng/ml. As shown in FIGS. 1A-1G, theaddition of as little as 0.3% manuka honey to cells in culture resultedin a significant decrease in the viability of B16.F1 cells (FIGS.1A-1C). This inhibitory effect on cell viability was dependent on bothmanuka honey concentration and total incubation time. By as early as 24hrs, the viabilities of B16.F1 cells cultured with manuka honey at finalconcentrations of 0.3, 0.6, 1.25 and 2.5% were 85%, 75%, 60% and 43% ofcontrol (no manuka honey) cultures, respectively (FIG. 1A).

In contrast, over the same time period, the viabilities of B16.F1 cellscultured in presence of 10 ng/ml or 50 ng/ml of the antineoplastic drugtaxol were reduced to 90% or 83% of control, respectively (FIG. 1A). Thedecreased cell viability was more pronounced as the time of cultureincreased to 48 hrs (FIG. 1B) or 72 hrs (FIG. 1C). At the latter timepoint, cell viability was reduced to 17% in cell cultures treated with2.5% manuka honey (FIG. 1C). Under the same conditions, cells culturedwith 10 ng/ml or 50 ng/ml taxol had a reduction in viability to 64% or34% of control, respectively. Essentially similar results were alsoobserved with the CT26 (FIGS. 1D-1E) and MCF-7 (FIGS. 1F-1G) cancer celllines. These results demonstrate that in vitro treatment of cancer cellswith low concentrations of manuka honey resulted in significantinhibition of cell proliferation.

Manuka Honey Induces Apoptosis in Cancer Cells

The potential mechanism by which manuka honey was causing decreased cellviability was addressed. Loss of cell membrane asymmetry, detectable byAnnexin V staining, represents one of the earliest events in apoptosis.B16.F1 cells were harvested at 24, 48, or 72 hours after treatment withdifferent concentrations of manuka honey (range 0.3% to 5.0% w/v) ortaxol (at a final concentration of 10 ng/ml), stained with AnnexinV-FITC and PI, and subjected to flow cytometric analysis. As can be seenin FIGS. 2A-2B, there was a dose-dependent, and time dependent, increasein the number of cells undergoing apoptosis (Annexin V-positive) afterculture with increasing concentrations of manuka honey. At 24 hr posttreatment, while the percent of Annexin V-positive cells in untreatedcontrol cultures was 1.0%, there were 1.5%, 11.8%, 13.7%, 14.5% and22.3% apoptotic cells after culture with 0.3%, 0.6%, 1.25%, 2.5% and5.0% manuka honey solution, respectively (left panels). In contrast,cells treated with taxol alone showed 32.8% apoptotic cells.Furthermore, in some cultures, a minor population of cells was observedto be positive for both Annexin V-FITC and PI, representing lateapoptotic cells. These cells amounted to 1.0-1.5% of total in cellcultures treated with manuka honey (at 0.6% concentration or higher) and3.9% in cells treated with taxol. The results of cell analysis followingsimilar treatments for 48 and 72 hrs (center and right panels,respectively) demonstrate a similar dose-dependent trend in apoptosis,with the overall levels of apoptotic cells being higher than thoseobserved at 24 hrs. For example, the percentage of total apoptotic cellsfollowing treatment with 0.3% manuka honey was 1.5%, 2.6% and 13.8%after 24, 48, and 72 hrs, respectively. The corresponding ratios of celldeath following treatment with 1.25% manuka honey suspension were 15.2%,27.9% and 35.8%, respectively. These findings suggest that the death ofcancer cells following exposure to low concentrations of manuka honeyoccurs via an apoptotic mechanism.

A critical component for the initiation of the apoptosis pathway is thesequential recruitment of a number of caspases leading to the activationof the effector caspase-3. This, in turn, leads to the cleavage of anumber of vital cellular substrates required for cell viability. Nextexplored was the mechanism of apoptosis induction in manukahoney-treated cancer cells. B16.F1 melanoma cells exposed to manukahoney (5% w/v final concentration) for 24 hrs exhibited a 24-foldincrease in caspase 3/7 activity (FIG. 3A). The induction of caspase 3/7activity in manuka honey treated cells was mainly due to activation ofcaspase-9 (FIG. 3C) but not caspase-8 (FIG. 3B).

In sharp contrast, treatment of the cells with paclitaxel (10 ng/ml) ledto a 2-fold increase in caspase 3/7 activity (FIG. 3A) and this wasassociated with a 2-fold increase in caspase-8 activity (FIG. 3B). Noevidence for induction of caspase-9 was observed in paclitaxel-treatedcancer cells (FIG. 3C). This implies that paclitaxel-induced cell deathoccurs mainly via the extrinsic pathway, which is in agreement withprevious observations. These findings demonstrate that manuka honeyactivates caspase-dependent apoptosis in cancer cells, a processinitiated through caspase-9, implicating the intrinsic pathway in manukahoney-induced cell death.

Bcl-2 is a member of a large family of cell survival-regulating proteinsconsisting of both proand anti-apoptotic regulators. Bcl-2 is apro-survival protein that acts upstream of the caspase pathway and, whenoverexpressed, can block cell apoptosis. Conversely, inhibition of Bcl-2protein expression predisposes to apoptosis. Therefore the level ofBcl-2 expression in B16.F1 cells following treatment with manuka honeyor taxol was determined. The results, shown in FIG. 4A, demonstratedecreased levels of Bcl-2 protein in manuka honey-treated cancer cells.In taxol-treated cells, Bcl-2 expression was substantially decreased by24 hr of culture and was undetectable by 72 hr. By contrast, in manukahoney-treated cancer cells, no decrease in Bcl-2 expression was observedat 24 hr; however, by 72 hr, there was >50% reduction in Bcl-2 levels.

One of the target proteins for active caspase-3 is the DNA repair enzymepoly(ADP-ribose) polymerase (or PARP). So, the effect of manuka honeytreatment on caspase-3 activation was investigated by Western blotanalysis using a monoclonal antibody against PARP that detects the fulllength (116 kD) and the cleaved (89 kD) forms of PARP (FIG. 4B). Lysatesof B16.F1 cells were prepared following treatment with manuka honey ortaxol for 24 hr (upper panels) or 72 hr (lower panels) and subjected toimmunoblot analysis with a PARP-specific antibody. After 24 hr ofculture, cleavage of PARP into the 89 kD fragment was evident only intaxol-treated cells (upper panel). However, after 72 hr, PARP wascleaved effectively in manuka honey-treated cells in a dose-dependentmanner (lower panel). Thus, at concentrations as low as 0.6%, manukahoney can effectively induce the caspase pathway leading to apoptosis ofcancer cells.

The effect of manuka honey-induced caspase activation on DNAfragmentation was also analyzed by agarose gel electrophoresis ofcellular DNA isolated after treatment. As shown in FIG. 4C, acharacteristic ladder pattern representing fragmented DNA was observedin cancer cells following treatment with manuka honey. At the highestmanuka honey concentration used (5.0%), the extent of DNA fragmentation,a classical apoptotic feature, was largely equivalent to that observedin taxol-treated cells. Taken together, the above results suggest thatmanuka honey leads to inhibition of cellular proliferation through areduction in pro-survival protein expression and activation of apoptosispathway.

In Vivo Toxicity Studies

Given the demonstrated in vitro effect of manuka honey on melanomacells, the potential of using manuka honey in an in vivo animal tumormodel was investigated. In preparation for that, a series of experimentswas carried out to test for any potential in vivo toxicity associatedwith intravenous administration of manuka honey. Mice received multiplei.v. injections of 50% (w/v) manuka honey solution diluted in sterilesaline for 3 weeks. At the end of this period, animals were sacrificedand blood was collected for hematological and clinical chemistryanalysis, the results of which are shown in FIGS. 5A-5F and 6A-6F,respectively. The findings demonstrated that multiple i.v. injections ofmanuka honey were not associated with any alterations in cellularconstituents of blood, including total WBC count, RBC count, plateletcount, % neutrophils, % lymphocytes and % monocytes (FIGS. 5A-5F).Furthermore, no significant changes were observed in the levels ofvarious chemical markers of organ dysfunction, including creatinine,BUN, AST, ALT, LDH, and glucose (FIG. 6A-6F).

Systemic Administration of Manuka Honey Inhibits Tumor Growth andEnhances Host Survival

The activity of manuka honey was evaluated in B16.F1 melanoma tumormodel. Mice were divided in four groups and treated by intravenousadministration (100-200 μl 2 times per week for up to 3 weeks) of manukahoney alone, at either 5%, 10% or 20% w/v, or saline as a control. Tumorvolume and animal survival were followed for up to 3 weeks posttreatment initiation. As shown in FIG. 7 the use of manuka honey aloneat higher concentrations (10% and 20%) was effective in retarding tumorgrowth and enhancing animal survival.

Systemic Manuka Honey Inhibits Tumor Growth and Enhances Host SurvivalWhen Used in Combination With Paclitaxel in a Melanoma Animal Model

The antitumor activity of manuka honey was evaluated in the syngeneicB16.F1 melanoma tumor model. C57BL/6 mice with established tumors(mean>50 mm3) were divided into four groups and treated by intravenousadministration (2 times per week for up to 3 weeks) of manuka honeyalone (50% w/v), taxol alone (10 mg/kg), manuka honey (50% w/v) plustaxol (10 mg/kg) or saline as a control. Tumor volume and animalsurvival were followed for up to 3 weeks post treatment initiation. Ascan be seen in FIG. 8A, tumor growth in saline-treated mice occurredcontinuously and rapidly, reaching a mean of 7035±516 mm3 by day 18 posttreatment, which corresponds to day 31 post tumor implantation. Micetreated with manuka honey alone exhibited a significant reduction intumor volume, with a mean of 4744±403 mm3, representing ˜33% inhibitionof tumor growth (p=0.0029). Mice treated with taxol alone or manukahoney plus taxol exhibited significantly greater degree of inhibition intumor growth, with mean tumor volumes being decreased by ˜61% comparedto control (p=<0.0001). Inhibition of tumor growth in taxol-treatedanimals was observed as early as 7 days after initiation of treatment,whereas manuka honey-treated mice exhibited a delay in tumor growthstarting on day 10 post treatment (FIG. 8A). The effect of the varioustreatments on animal survival was also followed (FIG. 8B). Mediansurvival for saline control group was ˜15 days and great majority ofmice (>80%) died by day 19 post treatment. In contrast, manukahoney-treated mice exhibited enhanced survival initially (shaded box inFIG. 8B) with an overall median survival of 19 days. By ˜3 weeks,however, their survival declined rapidly, and was ultimately comparableto saline controls at the end of the observation period (day 25 posttreatment). Similarly, paclitaxel-treated animals exhibited bettersurvival initially (median survival=20 days) but then declined reachingan overall survival of 20% at the end observation period. Lastly, miceco-treated with manuka honey plus taxol exhibited a marked enhancementin their overall survival with 55% of mice surviving (median >25 days),which was significantly different from controls (p=<0.0001). Takentogether, these findings demonstrate that intravenously-administeredmanuka honey has a modest, but significant, inhibitory effect on thegrowth of the highly tumorigenic B16.F1 melanoma cells with a transientimprovement in host survival. Moreover, when given in conjunction withan optimal dose of taxol, no additive or synergistic effect of manukahoney on overall tumor volume was observed. However, the combinationtreatment improved overall animal survival dramatically, suggestingperhaps a role for manuka honey in reducing drug-induced toxicity.

Further Studies on the Combination Treatment of Taxol at DifferentConcentrations and Manuka Honey Were Carried out on B16.F1 MelanomaTumor Model.

C57BL/6 mice with established tumors (mean>50 mm³) were divided intofour groups and treated by intravenous administration (2 times per weekfor up to 3 weeks) of manuka honey alone (50% w/v), taxol alone (5mg/kg), manuka honey (50% w/v) plus taxol (5 mg/kg) or saline as acontrol. Tumor volume and animal survival were followed for up to 3weeks post treatment initiation.

As shown in FIG. 9, administration of taxol at 5 mg/kg was ineffectiveat reducing tumor volume as compared to the use of saline. However whenadministered in combination with manuka honey, the combination of taxoland manuka honey was more effective at reducing tumor volume andenhancing survival than the control 14 days post-treatment.

In a further experiment the mice were divided into four groups andtreated by intravenous administration (2 times per week for up to 3weeks) of manuka honey alone (50% w/v), taxol alone (8 mg/kg), manukahoney (50% w/v) plus taxol (8 mg/kg) or saline as a control. Tumorvolume and animal survival were followed for up to 3 weeks posttreatment initiation. As shown in FIGS. 10A-10C, the combination oftaxol and manuka honey was shown to be effective in inhibiting tumorgrowth and enhancing survival than the control 14 days post treatment.

Combination In Vivo Treatment With Manuka Honey Plus Paclitaxel Leads toMarked Increase in Cancer Cell Death In Situ

Tumor tissue sections were prepared from tumors obtained from animalstreated with manuka honey, paclitaxel, manuka honey plus paclitaxel orsaline as control. Staining with caspase 3-specific mAb revealed thepresence of apoptotic cells, largely concentrated around the perimeterof necrotic tissue (FIGS. 11A-11D). By counting the number of caspase3-positive cells in a random selection of 10-20 high power fields (hpf),a quantitative estimate of apoptotic cell number could be achieved. Assummarized in FIG. 11E, the number of apoptotic cells in tumors ofuntreated mice was 3.6±0.4 per hpf. In mice treated with manuka honey ortaxol alone, the number of caspase 3-positive cells increased to10.1±1.0 or 11.7±1.8 per hpf, respectively. In contrast, there was afurther increase in the number of apoptotic cells observed in micetreated with taxol plus manuka honey, reaching a mean of 18.5±2.3 perhpf.

Systemic Administration of Manuka Honey Reverses Paclitaxel-InducedOrgan Toxicity

Groups of mice were injected with manuka honey, paclitaxel, manuka honeyplus paclitaxel or saline for a total period of 3 weeks (2 injectionsper week). The animals were then sacrificed and their organs (liver,spleen, and kidney) obtained and analyzed for the levels of glutathione(GSH), a major antioxidant effector. The levels of GSH in each organ areshown in FIGS. 12A-12C. The findings illustrate that paclitaxeltreatment results in a marked decrease in organ GSH levels, specially inthe liver and spleen. In sharp contrast, treatment with a combination ofmanuka honey plus paclitaxel largely reversed the decrease in organ GSHlevels. These results demonstrate unequivocally the ability of manukahoney to protect against the systemic toxicity induced by paclitaxel.

In the present study, a melanoma murine model known for its lowimmunogenicity, and hence high tumorigenicity, was used to demonstrate arole for manuka honey in retarding tumor growth in vivo. Histologicaland immunohistochemical evidence is provided to show that tumorretardation correlated with increased apoptosis of tumor cells. Moreintriguingly, the findings demonstrate that simultaneous treatment witha chemotherapeutic drug plus manuka honey led to a highly significantimprovement in overall animal survival. This suggests that the advantageof using intravenous manuka honey may well extend beyond its directantitumor activity to include the added beneficial effect of reducingchemotherapy drug-induced toxicity and enhancing host survival.

The main mechanism by which manuka honey appears to exert itsanti-proliferative effect on cancer cells is through the activation ofthe intrinsic apoptotic pathway, involving the induction of theinitiator caspase-9 which in turns activates the executioner caspase-3.In contrast, no evidence for the activation of caspase-8, and hence theextrinsic pathway, in manuka-treated cancer cells. In contrast,essentially the reverse was observed in taxol-treated cells wherecaspase-8, but not caspase-9, activation was evident. This is in linewith previous reports showing that taxol's effect on cell growth wasmediated mainly through the extrinsic apoptosis pathway without theinvolvement of caspase-9. Manuka honey-induced apoptosis is alsoassociated with the activation of PARP, induction of DNA fragmentationand loss of Bcl-2 expression. The results of the in vitro cell viabilitystudies demonstrate that manuka honey was effective against severaltypes of murine and human cancer cell lines at very low concentrations.The IC50 values (manuka concentrations required for 50% inhibition ofcell growth) of the murine B16.F1 melanoma cells, calculated after 24,48 or 72 hrs of exposure to manuka honey were 2%, 1.3% and 0.8%,respectively. Similarly, for CT26 cells, the IC50 values at 24 and 72hrs were 2% and 1%. Interestingly, the observed IC50 values for MCF-7cells are significantly higher, calculated to be >5% and 4% manuka honeyat 24 and 72 hr, respectively. The observed relative resistance of theMCF-7 cells to manuka honey-induced apoptosis may well be due to thefact that these cells are known to be deficient in caspase-3 expression(Janicke R U (2009) Breast Cancer Res Treat 117: 219-221, Janicke R U etal (1998) J Biol Chem 273: 9357-9360). Nevertheless, it is intriguing tohypothesize that the fact that manuka honey could still induce apoptosisin caspase-3-deficient cells may well indicate that a secondary pathwaycould also function, albeit at reduced efficiency.

The ability of manuka honey, at concentrations as little as 0.3-0.6%,were found to induce apoptosis in cancer cells. This was demonstratedusing several approaches, including cell viability and flowcytometricassays, direct determination of increased caspase 3/7 and 9 enzymeactivities, and DNA fragmentation. Moreover, using a syngeneic melanomamodel, it was demonstrated that manuka honey was also effective againstcancer cells in vivo, as evidenced by the observed decrease in tumorvolume and increased apoptosis of tumor cells detected by caspase-3immunohistochemical analysis. Although detailed analyses of the effectof other types of honey on cancer cells remain to be done, based on thecell viability data, the results suggest that manuka honey may besuperior in its anticancer potential than other types of honey. UsingTualang honey, Ghashm and coworkers reported IC50 values of 3.5-4.0%against human oral squamous cell carcinoma and osteosarcoma cell lines.Swellam et al also reported IC50 values of 2.0-4.0% against bladdercancer cell lines using unfractionated honey from Manitoba, Japan Thesedifferences are most likely due to variations in honey content,particularly in polyphenols and phenolic acids with known antitumoractivities. Scientific evidence for the use of honey in wound healinghas been accumulating over the past few years, largely as a result ofcompleted small-scale clinical trials. Many properties of honey havebeen described that aid the process of wound healing such as activatingthe innate immune system, inducing the migration of neutrophils andmacrophages, promoting the debridement of devitalized tissue,stimulating angiogenesis and granulation, and preventing infection.Manuka honey has the capacity to stimulate macrophages to release innateimmune mediators, such as TNF-α, IL-1β and IL-6, which are essential fortissue healing and for limiting microbial infections.

The present invention has found that there is a beneficial effect ofadministering manuka honey together with taxol. In comparison with theanimal group receiving taxol alone, those treated with taxol and manukahoney exhibited a highly significant improvement in survival. Thisoccurred despite having almost identical mean tumor volumes in bothexperimental groups, suggesting there was no added or synergistic actionof both agents on inhibiting tumor growth, at least at the optimal doseof taxol used in this study. These findings suggest that manuka honeyadministration may decrease the toxic side effects of chemotherapeuticdrugs. Support for this hypothesis is evident in recently publishedreports demonstrating potent antiinflammatory, antioxidant and cellgrowth-promoting activities among various types of honey, includingmanuka honey. Moreover, intravenous administration of honey protectedagainst organ failure in rabbits following LPS-induced sepsis throughthe inhibition of inflammation and myeloperoxidase production. Thus,manuka honey may well improve survival of taxol treated, tumor-bearing,mice via a similar protective mechanism. The current findings shouldfacilitate further work to examine whether manuka honey could synergizewith, or be a substitute for, chemotherapeutic drugs given atsub-optimal doses for cancer therapy.

Manuka honey (MH)/Paclitaxel combination has unique properties forlimiting toxicities associated with administration of chemotherapy drugsthat are distinct from sugar/Paclitaxel solution.

An additional study was conducted in which the effect of administeringPaclitaxel/dextrose solution (50% w/v) vs. Paclitaxel/manuka honey (50%w/v) MH solution into mice was investigated.

The injections were carried out following exactly the same protocoldescribed hereinabove (2 injections per week, for a total of 3 weeks).The only difference being that the chemotherapeutic agent was preparedeither in dextrose or in MH solution. At the end of treatment, theexpression of oxidative stress and anti-oxidant genes in the livers oftreated mice were anelized. For this purpose, use was made of a highlyreliable and sensitive gene expression profiling PCR array usingreal-time PCR, the assay, called RT Profiler™ PCR Array Mouse OxidativeStress and Antioxidant Defense. The RT Profiler™, was purchased fromQiagen and allowed the examination of expression profiles of a total of84 different genes involved in inflammation and oxidative stresspathways.

More specifically, the results of this analysis revealed that as many as37 genes of a total of 84 (44%) had significantly different levels ofexpression between the two treatment groups. As shown in FIG. 25 of thedrawings, out of the 37 differentially expressed genes, 27 genes weresignificantly over-expressed and 10 genes were under-expressed in thelivers of Paclitaxel/MH-treated mice compared toPaclitaxel/dextrose-treated mice. These findings refute any suggestionthat the two treatments are similar or comparable in their effect.

The data presented in FIG. 25 proves that administration of achemotherapeutic drug made up in MH solution leads to significantlyaltered expression of various genes involved in the oxidative stresspathway compared to giving the same drug in dextrose solution. It wasdecided to test this particular pathway because it is well known thatchemotherapy drugs have substantial toxic side-effects on the host andthis will activate the oxidative stress response. Moreover, theseresults assist in the understanding of how the reduction in systemictoxicity is achieved by MH.

Additional In Vitro Tumor Treatment Studies Using Combination ofpaclitaxel (at doses of 5 or 8 mg/kg body weight) and Manuka Honey (MH)

Materials & Methods for Additional In Vivo Tumor Studies

One syngeneic tumor model was used in this study, namely the B16.F1melanoma (skin cancer) model. The B16.F1 cancer cells originated inC57BL/6 mice. The procedure for tumor implantation is briefly asfollows, C57BL/6 (8-10 mice per group) were inoculated subcutaneously inthe right flank with 2×10⁵ B16.F1 melanoma cells and staged to day12-14.

Tumor growth was followed by quantitative determination of tumor volume,measured as the product of the perpendicular diameters using digitalcalipers, according to the formula: volume=L×W²/2. For the B16.F1 model,once tumors were established, mice were given intravenous (i.v.) doses(100 μl per injection) of (1) saline (control), (2) 50% (w/v) manukahoney (MH) suspension in sterile saline, (3) paclitaxel (at doses of 5or 8 mg/kg body weight) or (4) MH (50% w/v)+paclitaxel. All treatmentswere routinely administered twice per week, using freshly preparedreagents. Tumor growth and animal survival were followed for thesubsequent 50 days.

The results of the study are summarised below under the headings“Results#1” and “Results #2”.

Results #1

FIGS. 13 and 14 show the results of the in vitro tumor study and showsthat combination therapy for melanoma is more potent than the individualtreatments with either taxol or MH alone. In this study, the dose ofpaclitaxel used was 5 mg/kg. As shown in FIG. 13, treatment ofmelanoma-bearing mice with a low dose of paclitaxel (5 mg/kg) alone orMH alone had no effect of tumor growth. However, combination treatmentled to a significant inhibition in tumor size, which was evident at days14, 17 and 21 after start of treatment (see FIG. 13). At day 21 oftreatment, the mean tumor size in the combination treatment group was42% lower than in control saline-treated mice (FIG. 14). Asterisks inFIG. 13 denote statistically significant differences between theindicated experimental groups (**, p<0.01; *, p<0.05).

As shown in FIG. 14, based on statistical analysis, neither paclitaxelor MH treatment alone resulted in any significant alterations in tumorgrowth.

In sharp contrast to the combined expected cumulative effect oftreatment with paclitaxel (5 mg/kg) alone plus treatment with maukahoney (50% w/v) alone, the results shown in FIG. 14 clearly demonstratesthat on day 21 in mice treated with paclitaxel (5 mg/kg) and mauka honey(50% w/v), the reduction in tumor growth is, contrary to allexpectations, a significant reduction of 3656 mm³ in tumor volume. Moreparticularly, the observed reduction (˜42%) in tumor growth in animalstreated with MH+paclitaxel is statistically significant (p<0.01).

This finding is surprising and significant and indicates that treatmentwith the combination of paclitaxel (5 mg/kg) and mauka honey (50% w/v)results in tumor reduction which is much higher than the expectedbecause it is significantly and markedly higher than the cumulative sumof the tumor volume reduction shown in mice treated with paclitaxel (5mg/kg) alone plus mauka honey (50% w/v) alone.

FIGS. 15 and 16 show the combination treatment of mauka honey (50% w/v)plus paclitaxel (5 mg/kg) also leads to a significant improvement inoverall animal survival compared to the control group. Moreparticularly, compared to the control, untreated, mice, treatment withMH alone or paclitaxel (5 mg/kg) alone resulted in no significantdifference in animal survival. In contrast, treatment with thecombination of MH plus paclitaxel (5 mg/kg) led to 50% survival oftumor-bearing mice. Asterisks denote statistically significantdifferences between the indicated experimental group in comparison tocontrol group (*, p<0.05).

Results #2

FIGS. 17 and 18, summarize the data from the studies using paclitaxel at8 mg/kg. In this scenario, treatment manuka honey (50% w/v) (MH) aloneand paclitaxel 8 mg/kg alone had an intermediate effect on reducingtumor growth but the combination treatment of paclitaxel at 8 mg/kg andmauka honey (50% w/v) was again significantly superior in bothcontrolling tumor growth and animal survival. Overall animal survivalwas best in animals treated with the combination of mauka honey (50%w/v) and paclitaxel 8 mg/kg (64% survival compared to 36% withpaclitaxel 8 mg/kg alone). Asterisks denote statistically significantdifferences between the indicated experimental groups (**, p<0.01; *,p<0.05).

More particularly, as shown in FIG. 18, the sum of the reduction of thetumor volume in the case of treatment with paclitaxel (8 mg/kg) aloneplus the reduction of the tumor volume in the case of treatment withmanuka honey 50% w/v alone shows an expected combined reduction of 3750mm³. In sharp contrast, FIG. 18 shows that the reduction of tumor volumeat day 17 in mice treated with manuka honey 50% w/v and paclitaxel (8mg/kg) is 4275 mm³.

This finding is surprising and significant and demonstrates that thereduction in tumor volume of mice treated with a combination ofpaclitaxel (8 mg/kg) and manuka honey (50% w/v) is significantly andmarkedly higher than the sum of the reduction in tumor volume in micetreated with manuka honey (50% w/v) only and paclitaxel (8 mg/kg) only.These findings further indicate that the combination of paclitaxel (8mg/kg) and manuka honey (50% w/v) is more than the cumulative effect ofthe ingredients alone, i.e., more than the sum of the individual effectof the manuka honey (50% w/v) only and paclitaxel (8 mg/kg) administeredalone.

Furthermore, the data shown in FIG. 20 indicates that the percentagesurvival for the sum of the percentage survival of mice treated withpaclitaxel (8 mg/kg) alone plus manuka honey (50% w/v) alone is 54.6%(=36.4%+18.2%). In sharp contrast, the percentage of survival of micetreated with a combination of paclitaxel (8 mg/kg) and manuka honey (50%w/v) is 63.4%, i.e., is markedly and significantly higher than thepercentage survival for the sum of the mice treated with paclitaxel (8mg/kg) alone plus manuka honey (50% w/v) alone.

In Vitro Tumor Treatment Studies Using Combination of Oxaliplatin andManuka Honey (50% w/v) (MH)

Materials & Methods for In Vivo Tumor Studies

Two different syngeneic tumor models were used in this study, namely,MC38 colorectal cancer and 4T1 metastatic breast adenocarcinoma.

MC38 cancer cells originated in C57BL/6 mice while 4T1 cells originatedin BALB/c mice. The procedure for tumor implantation is briefly asfollows, C57BL/6 (8-10 mice per group) were inoculated subcutaneously inthe right flank with 2×10⁵ B16.F1 melanoma cells or 1×10⁵ MC38 coloncancer cells, and staged to day 12-14. For the 4T1 breast cancer cells,BALB/c mice were implanted orthotopically in the mammary fat pad with1×10⁶ cancer cells/mouse.

Tumor growth was followed by quantitative determination of tumor volume,measured as the product of the perpendicular diameters using digitalcalipers, according to the formula: volume=L×W²/2.

For the colon and breast cancer models, mice bearing tumors were treatedwith (1) saline (control), (2) manuka honey (50% w/v), (3) Oxaliplatinat 6 mg/kg or (4) manuka honey (50% w/v)+Oxaliplatin (6 mg/kg). Themanuka honey (50% w/v) was given intravenously (i.v.) twice per week.Oxaliplatin 6 mg/kg was administered intraperitoneally (i.p.) once perweek. The total treatment period was 3 weeks.

The results of the study are summarised below under the headings“Results #3” and

“Results #4”.

Results #3

Referring to the data presented in FIGS. 21 and 22, the data shows thatcombination treatment with manuka honey (50% w/v) plus Oxaliplatin at 6mg/kg body weight is effective in inhibiting colon cancer inexperimental mouse models.

The results presented in FIGS. 21 and 22 show that combining manukahoney (50% w/v) with Oxaliplatin at 6 mg/kg body weight, leads to a moreeffective inhibition of colon cancer growth and a significantenhancement in host survival (up to 40% animal survival compared to 0%in the case of treatment with saline, Oxaliplatin at 6 mg/kg alone andmanuka honey 50% w/v).

The dosing regimen for the different treatments is also shown in FIG.21. Asterisks denote statistically significant differences between theindicated experimental group in comparison to control group (*, p<0.05).

More specifically, as can be seen from FIGS. 21 and 22, at day 50, thepercentage survival of mice treated with saline, Oxaliplatin (6 mg/kg)and manuka honey alone (50% w/v) is zero. In sharp contrast, at day 50,survival of mice treated with a combination of Oxaliplatin (6 mg/kg) andmanuka honey (50% w/v) was as high as 40%.

This finding is surprising and significant and demonstrates that thepercentage survival of mice treated with a combination of Oxaliplatin (6mg/kg) and manuka honey (50% w/v) is significantly and markedly higherthan the sum of the percentage survival of mice treated with manukahoney (50% w/v) only and Oxaliplatin (6 mg/kg) only. These findingsfurther indicate that the combination of Oxaliplatin (6 mg/kg) andmanuka honey (50% w/v) is more than the cumulative effect of theingredients alone, i.e., more than the sum of the individual effect ofthe manuka honey (50% w/v) only and Oxaliplatin (6 mg/kg) administeredalone.

Results #4

Referring to the data presented in FIGS. 23 and 24, the data shows thatcombination treatment with manuka honey (50% w/v) plus Oxaliplatin at 6mg/kg body weight is effective in inhibiting metastatic breast cancer inexperimental mouse models.

More particularly, similar findings were obtained using the combinationtreatment of MH (50% w/v) and Oxaliplatin at 6 mg/kg body weight in ametastatic breast cancer model in mice. In this model, the use ofOxaliplatin at 6 mg/kg body weight alone had only a marginal effect oncancer growth and animal survival. However, as shown in FIGS. 23 and 24,the combination treatment with MH (50% w/v) and Oxaliplatin at 6 mg/kgbody weight resulted in significant improvement in both reduction ofcancer growth and animal survival. From the results shown in FIGS. 23and 24 it is clear that the combination of MH (50% w/v) and Oxaliplatinat 6 mg/kg body weight had significantly improved outcomes that weredemonstrably more than the combined effect of the individual agentsalone. Asterisks denote statistically significant differences betweenthe indicated experimental group in comparison to control group (*,p<0.05).

More specifically, as can be seen from FIGS. 23 and 24, at day 50, thepercentage survival of mice treated with saline was zero, whereas, only22% of mice treated with Oxaliplatin (6 mg/kg) alone survived and only11% of mice treated with manuka honey alone (50% w/v) survived.

In sharp contrast, at day 50, mice treated with a combination ofOxaliplatin (6 mg/kg) and manuka honey (50% w/v) was as high as 46%.This finding is surprising and significant and demonstrates that thepercentage survival of mice treated with a combination of Oxaliplatin (6mg/kg) and manuka honey (50% w/v) is significantly and markedly higherthan the sum of the percentage survival of mice treated with manukahoney (50% w/v) only and Oxaliplatin (6 mg/kg) only. These findingsfurther indicate that the combination of Oxaliplatin (6 mg/kg) andmanuka honey (50% w/v) is more than the cumulative effect of theingredients alone, i.e., more than the sum of the individual effect oftreatment with the manuka honey (50% w/v) only and Oxaliplatin (6 mg/kg)administered alone.

1. An intravenous pharmaceutical composition comprising: a diluent; atleast 50% w/v of manuka honey diluted by the diluent; and at least onechemotherapeutic agent, wherein the chemotherapeutic agent is selectedfrom the group consisting of paclitaxel between about 5 mg/kg of apatient to be treated and 10 mg/kg of a patient to be treated;oxaliplatin at least about 1 mg/kg of a patient to be treated; and anypharmaceutically acceptable salts thereof.
 2. The pharmaceuticalcomposition of claim 1, wherein the chemotherapeutic agent is selectedfrom the group consisting of paclitaxel 5 mg/kg of a patient to betreated; paclitaxel 8 mg/kg of a patient to be treated; oxaliplatin 6mg/kg of a patient to be treated; and any pharmaceutically acceptablesalts thereof.
 3. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition is in the form of a solution.
 4. Thepharmaceutical composition of claim 1, wherein the diluent is a sterilesaline solution.
 5. A method of treating skin cancer, colon cancer,colorectal cancer and/or breast cancer in a patient comprisingadministering to the patient a pharmaceutically acceptable amount of thecomposition as claimed in claim
 1. 6. The method of claim 5, wherein themanuka honey and chemotherapeutic agent are administered sequentially.7. The method of claim 5, wherein the manuka honey and chemotherapeuticagent are administered simultaneously.
 8. The method of claim 5, furthercomprising administered a second chemotherapeutic agent.
 9. The methodof claim 8, wherein the second chemotherapeutic agent is selected fromthe group consisting of cisplatin, doxorubicin, trastuzumab, and anypharmaceutically acceptable salts thereof.
 10. The method of claim 5,wherein the method is for enhancing the survival of the patient.
 11. Themethod of claim 5, wherein the method is for reducing the toxicity ofsaid at least one chemotherapeutic agent in the patient.
 12. The methodof claim 8, wherein the method is for reducing the toxicity of thesecond chemotherapeutic agent.
 13. A unit dose or multi-dose containercontaining the intravenous pharmaceutical composition as claimed inclaim 1.